New insights into the classification of the RAC1 P29S hotspot mutation in melanoma as an oncogene

New insights into the classification of the RAC1 P29S hotspot mutation in melanoma as an oncogene | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article New insights into the classification of the RAC1 P29S hotspot mutation in melanoma as an oncogene Mohammad reza Ahmadian, Amin Mirzaiebadizi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6196528/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Oct, 2025 Read the published version in Cancer Gene Therapy → Version 1 posted You are reading this latest preprint version Abstract The RAC1 P29S hotspot mutation, prevalent in melanoma, drives tumorigenesis by enhancing molecular interactions and hyperactivating key signaling pathways, making it a compelling target for cancer therapy. This study provides a comprehensive biochemical characterization of RAC1 P29S compared to wild-type RAC1 and mutations T17N and F28L. The P29S mutation significantly impairs nucleotide binding to guanosine triphosphate (GTP) and guanosine diphosphate, accelerating intrinsic nucleotide exchange. While minimally affecting regulation by guanosine dissociation inhibitor 1, RAC1 P29S exhibits reduced activation via diffuse B-cell lymphoma family guanine nucleotide exchange factors but retains effective activation by dedicator of cytokinesis 2. Critically, the P29S mutation severely impairs GTPase-activating protein-stimulated GTP hydrolysis, most likely contributing to RAC1 P29S hyperactivation by prolonging its GTP-bound form. RAC1 P29S displays a stronger binding affinity for IQ motif-containing GTPase-activating protein 1 than for p21-activated kinase 1, highlighting the role of the former in scaffolding RAC1 P29S -driven signaling. In serum-starved cells, RAC1 P29S predominantly adopts an active GTP-bound state. RAC1 P29S overexpression activates key cancer-associated pathways, including extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, reinforcing its role as an oncogenic driver in melanoma. These insights suggest potential therapeutic targets for melanoma treatment, including RAC1 regulators and modulators. Biological sciences/Biochemistry/Proteomics Biological sciences/Molecular biology/Proteomics Small GTPases RAC1 P29S mutation oncogene gain of function melanoma drug resistance DOCK2 p50GAP IQGAP1 Full Text Additional Declarations There is NO conflict of interest to disclose. Supplementary Files MirzaiebadiziRAC1P29SSupplementaryInformation.pdf Supplemetal Information Cite Share Download PDF Status: Published Journal Publication published 01 Oct, 2025 Read the published version in Cancer Gene Therapy → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6196528","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":428452705,"identity":"0a4eafa2-09a4-4ed2-b71d-95fac93b4340","order_by":0,"name":"Mohammad reza Ahmadian","email":"data:image/png;base64,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","orcid":"","institution":"Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University","correspondingAuthor":true,"prefix":"","firstName":"Mohammad","middleName":"reza","lastName":"Ahmadian","suffix":""},{"id":428452706,"identity":"401ec8bb-1dd0-4ac2-8ca4-19aed5f7dedf","order_by":1,"name":"Amin Mirzaiebadizi","email":"","orcid":"https://orcid.org/0000-0002-3340-586X","institution":"Institute of Biochemistry and Molecular Biology II, Medical Faculty and University Hospital D\u0026#x00FC","correspondingAuthor":false,"prefix":"","firstName":"Amin","middleName":"","lastName":"Mirzaiebadizi","suffix":""}],"badges":[],"createdAt":"2025-03-10 14:36:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6196528/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6196528/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41417-025-00965-x","type":"published","date":"2025-10-01T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":92638468,"identity":"5a7d61c6-4256-467b-9cf3-7e20ea0ce77f","added_by":"auto","created_at":"2025-10-02 07:11:48","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2132967,"visible":true,"origin":"","legend":"Article File","description":"","filename":"MirzaiebadiziRAC1P29SArticlefile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6196528/v1_covered_0c2ce4d2-9a6a-41fa-9776-e9abdc48ecd2.pdf"},{"id":79351146,"identity":"7d48bb7c-1702-4aa0-aad6-da3e102202a5","added_by":"auto","created_at":"2025-03-27 10:24:32","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10053974,"visible":true,"origin":"","legend":"Supplemetal Information","description":"","filename":"MirzaiebadiziRAC1P29SSupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6196528/v1/6943ee997945359716b970dc.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"New insights into the classification of the RAC1 P29S hotspot mutation in melanoma as an oncogene","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-gene-therapy","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cgt","sideBox":"Learn more about [Cancer Gene Therapy](http://www.nature.com/cgt/)","snPcode":"41417","submissionUrl":"https://mts-cgt.nature.com/cgi-bin/main.plex","title":"Cancer Gene Therapy","twitterHandle":"@cgtnature","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Small GTPases, RAC1, P29S mutation, oncogene, gain of function, melanoma, drug resistance, DOCK2, p50GAP, IQGAP1","lastPublishedDoi":"10.21203/rs.3.rs-6196528/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6196528/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe RAC1\u003csup\u003eP29S \u003c/sup\u003ehotspot mutation, prevalent in melanoma, drives tumorigenesis by enhancing molecular interactions and hyperactivating key signaling pathways, making it a compelling target for cancer therapy. This study provides a comprehensive biochemical characterization of RAC1\u003csup\u003eP29S\u003c/sup\u003e compared to wild-type RAC1 and mutations T17N and F28L. The P29S mutation significantly impairs nucleotide binding to guanosine triphosphate (GTP) and guanosine diphosphate, accelerating intrinsic nucleotide exchange. While minimally affecting regulation by guanosine dissociation inhibitor 1, RAC1\u003csup\u003eP29S\u003c/sup\u003e exhibits reduced activation via diffuse B-cell lymphoma family guanine nucleotide exchange factors but retains effective activation by dedicator of cytokinesis 2. Critically, the P29S mutation severely impairs GTPase-activating protein-stimulated GTP hydrolysis, most likely contributing to RAC1\u003csup\u003eP29S\u003c/sup\u003e hyperactivation by prolonging its GTP-bound form. RAC1\u003csup\u003eP29S\u003c/sup\u003e displays a stronger binding affinity for IQ motif-containing GTPase-activating protein 1 than for p21-activated kinase 1, highlighting the role of the former in scaffolding RAC1\u003csup\u003eP29S\u003c/sup\u003e-driven signaling. In serum-starved cells, RAC1\u003csup\u003eP29S\u003c/sup\u003e predominantly adopts an active GTP-bound state. RAC1\u003csup\u003eP29S\u003c/sup\u003e overexpression activates key cancer-associated pathways, including extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, reinforcing its role as an oncogenic driver in melanoma. These insights suggest potential therapeutic targets for melanoma treatment, including RAC1 regulators and modulators.\u003c/p\u003e","manuscriptTitle":"New insights into the classification of the RAC1 P29S hotspot mutation in melanoma as an oncogene","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-27 10:16:27","doi":"10.21203/rs.3.rs-6196528/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-gene-therapy","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cgt","sideBox":"Learn more about [Cancer Gene Therapy](http://www.nature.com/cgt/)","snPcode":"41417","submissionUrl":"https://mts-cgt.nature.com/cgi-bin/main.plex","title":"Cancer Gene Therapy","twitterHandle":"@cgtnature","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"986dad97-8879-43b4-8252-d9dca7f7b9d7","owner":[],"postedDate":"March 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45648197,"name":"Biological sciences/Biochemistry/Proteomics"},{"id":45648198,"name":"Biological sciences/Molecular biology/Proteomics"}],"tags":[],"updatedAt":"2025-10-02T07:11:41+00:00","versionOfRecord":{"articleIdentity":"rs-6196528","link":"https://doi.org/10.1038/s41417-025-00965-x","journal":{"identity":"cancer-gene-therapy","isVorOnly":false,"title":"Cancer Gene Therapy"},"publishedOn":"2025-10-01 04:00:00","publishedOnDateReadable":"October 1st, 2025"},"versionCreatedAt":"2025-03-27 10:16:27","video":"","vorDoi":"10.1038/s41417-025-00965-x","vorDoiUrl":"https://doi.org/10.1038/s41417-025-00965-x","workflowStages":[]},"version":"v1","identity":"rs-6196528","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6196528","identity":"rs-6196528","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}